Adenosine diphosphate (ADP) and adenosine 3'5'-cyclic monophosphate (cAMP) respectively play major roles in platelet activation and inhibiton. ADP induces human platelets to change shape from discs to spiculated spheres, followed by platelet aggregation and storage granule secretion. ADP, a charged compound, does not enter the cell, but reacts reversibly with a finite number of receptor sites. Identification and characterization of such a membrane protein is crucial for understanding platelet activation and might make possible a rational approach to inhibiting thrombus formation. Elevation of platelet cAMP leads to inhibition of platelet shape change, adhesion, aggregation, and release of granule contents. PGI2 and adenosine elevate cAMP by stimulating adenylate cyclase while inhibitors of cAMP phosphodiesterases (PDE), such as dipyridamole, enhance the elevation. Little is known about the various cAMP PDE in platelets. We propose to further characterize and study the regulation of the low K-m cAMP PDE and to isolate and purify additional cyclic nucleotide PDE, including the cGMP stimulated PDE and the cGMP PDE. We will characterize the enzymes in regard to size, pI pH optimum, subunit structure, metal cofactor requirements, kinetic behavior with both cAMP and cGMP, regulation by cAMP and proteolysis, as well as the action of pharmacologic inhibitors. We will also use an affinity analogue 2-[(4-bromo-2,3, dioxobutyl)thio] adenosine cyclic 3'5' monophosphate (2-BDB-T-cAMP) to study the kinetics of inactivation of cAMP PDE's and to identify the active site residues responsible. Finally, we will identify and purify an ADP binding protein of platelets labeled by FSBA. We will establish the peptides and/or amino acids modified by FSBA. We will investigate to define the functional consequences of the binding, including interactions with other receptors, as well as the relationship of the ADP receptor to known platelet membrane glycoproteins. These studies should yield new knowledge of nucleotide binding proteins such as an ADP receptor and cAMP PDE which should make possible the rational development of drugs to modify platelet responses in thrombotic disorders.